Validation of a Low-Cost Electrocardiography (ECG) System for Psychophysiological Research.

BACKGROUND AND OBJECTIVE: The reliability of low-cost mobile systems for recording Electrocardiographic (ECG) data is mostly unknown, posing questions regarding the quality of the recorded data and the validity of the extracted physiological parameters. The present study compared the BITalino toolkit with an established medical-grade ECG system (BrainAmp-ExG). METHODS: Participants underwent simultaneous ECG recordings with the two instruments while watching pleasant and unpleasant pictures of the "International Affective Picture System" (IAPS). Common ECG parameters were extracted and compared between the two systems. The Intraclass Correlation Coefficients (ICCs) and the Bland-Altman Limits of Agreement (LoA) method served as criteria for measurement agreement. RESULTS: All but one parameter showed an excellent agreement (>80%) between both devices in the ICC analysis. No criteria for Bland-Altman LoA and bias were found in the literature regarding ECG parameters. CONCLUSION: The results of the ICC and Bland-Altman methods demonstrate that the BITalino system can be considered as an equivalent recording device for stationary ECG recordings in psychophysiological experiments.

Maize Brittle Stalk2-Like3, encoding a COBRA protein, functions in cell wall formation and carbohydrate partitioning.

Carbohydrate partitioning from leaves to sink tissues is essential for plant growth and development. The maize (Zea mays) recessive carbohydrate partitioning defective28 (cpd28) and cpd47 mutants exhibit leaf chlorosis and accumulation of starch and soluble sugars. Transport studies with 14C-sucrose (Suc) found drastically decreased export from mature leaves in cpd28 and cpd47 mutants relative to wild-type siblings. Consistent with decreased Suc export, cpd28 mutants exhibited decreased phloem pressure in mature leaves, and altered phloem cell wall ultrastructure in immature and mature leaves. We identified the causative mutations in the Brittle Stalk2-Like3 (Bk2L3) gene, a member of the COBRA family, which is involved in cell wall development across angiosperms. None of the previously characterized COBRA genes are reported to affect carbohydrate export. Consistent with other characterized COBRA members, the BK2L3 protein localized to the plasma membrane, and the mutants condition a dwarf phenotype in dark-grown shoots and primary roots, as well as the loss of anisotropic cell elongation in the root elongation zone. Likewise, both mutants exhibit a significant cellulose deficiency in mature leaves. Therefore, Bk2L3 functions in tissue growth and cell wall development, and this work elucidates a unique connection between cellulose deposition in the phloem and whole-plant carbohydrate partitioning.

Genetic variation among 481 diverse soybean accessions, inferred from genomic re-sequencing.

We report characteristics of soybean genetic diversity and structure from the resequencing of 481 diverse soybean accessions, comprising 52 wild (Glycine soja) selections and 429 cultivated (Glycine max) varieties (landraces and elites). This data was used to identify 7.8 million SNPs, to predict SNP effects relative to genic regions, and to identify the genetic structure, relationships, and linkage disequilibrium. We found evidence of distinct, mostly independent selection of lineages by particular geographic location. Among cultivated varieties, we identified numerous highly conserved regions, suggesting selection during domestication. Comparisons of these accessions against the whole U.S. germplasm genotyped with the SoySNP50K iSelect BeadChip revealed that over 95% of the re-sequenced accessions have a high similarity to their SoySNP50K counterparts. Probable errors in seed source or genotype tracking were also identified in approximately 5% of the accessions.

Glyco-engineered HEK 293-F cell lines for the production of therapeutic glycoproteins with human N-glycosylation and improved pharmacokinetics.

N-glycosylated proteins produced in human embryonic kidney 293 (HEK 293) cells often carry terminal N-acetylgalactosamine (GalNAc) and only low levels of sialylation. On therapeutic proteins, such N-glycans often trigger rapid clearance from the patient's bloodstream via efficient binding to asialoglycoprotein receptor (ASGP-R) and mannose receptor (MR). This currently limits the use of HEK 293 cells for therapeutic protein production. To eliminate terminal GalNAc, we knocked-out GalNAc transferases B4GALNT3 and B4GALNT4 by CRISPR/Cas9 in FreeStyle 293-F cells. The resulting cell line produced a coagulation factor VII-albumin fusion protein without GalNAc but with increased sialylation. This glyco-engineered protein bound less efficiently to both the ASGP-R and MR in vitro and it showed improved recovery, terminal half-life and area under the curve in pharmacokinetic rat experiments. By overexpressing sialyltransferases ST6GAL1 and ST3GAL6 in B4GALNT3 and B4GALNT4 knock-out cells, we further increased factor VII-albumin sialylation; for ST6GAL1 even to the level of human plasma-derived factor VII. Simultaneous knock-out of B4GALNT3 and B4GALNT4 and overexpression of ST6GAL1 further lowered factor VII-albumin binding to ASGP-R and MR. This novel glyco-engineered cell line is well-suited for the production of factor VII-albumin and presumably other therapeutic proteins with fully human N-glycosylation and superior pharmacokinetic properties.

Early Changes in Pain Acceptance Predict Pain Outcomes in Interdisciplinary Treatment for Chronic Pain.

Studies have shown that pain acceptance is associated with a better pain outcome. The current study explored whether changes in pain acceptance in the very early treatment phase of an interdisciplinary cognitive-behavioral therapy (CBT)-based treatment program for chronic pain predict pain outcomes. A total of 69 patients with chronic, non-malignant pain (at least 6 months) were treated in a day-clinic for four-weeks. Pain acceptance was measured with the Chronic Pain Acceptance Questionnaire (CPAQ), pain outcomes included pain intensity (Numeric Rating Scale, NRS) as well as affective and sensory pain perception (Pain Perception Scale, SES-A and SES-S). Regression analyses controlling for the pre-treatment values of the pain outcomes, age, and gender were performed. Early changes in pain acceptance predicted pain intensity at post-treatment measured with the NRS (B = -0.04 (SE = 0.02); T = -2.28; p = 0.026), affective pain perception at post-treatment assessed with the SES-A (B = -0.26 (SE = 0.10); T = -2.79; p = 0.007), and sensory pain perception at post-treatment measured with the SES-S (B = -0.19 (SE = 0.08); T = -2.44; p = 0.017) . Yet, a binary logistic regression analysis revealed that early changes in pain acceptance did not predict clinically relevant pre-post changes in pain intensity (at least 2 points on the NRS). Early changes in pain acceptance were associated with pain outcomes, however, the impact was beneath the threshold defined as clinically relevant.

Maize Carbohydrate Partitioning Defective33 Encodes an MCTP Protein and Functions in Sucrose Export from Leaves.

To sustain plant growth, development, and crop yield, sucrose must be transported from leaves to distant parts of the plant, such as seeds and roots. To identify genes that regulate sucrose accumulation and transport in maize (Zea mays), we isolated carbohydrate partitioning defective33 (cpd33), a recessive mutant that accumulated excess starch and soluble sugars in mature leaves. The cpd33 mutants also exhibited chlorosis in the leaf blades, greatly diminished plant growth, and reduced fertility. Cpd33 encodes a protein containing multiple C2 domains and transmembrane regions. Subcellular localization experiments showed the CPD33 protein localized to plasmodesmata (PD), the plasma membrane, and the endoplasmic reticulum. We also found that a loss-of-function mutant of the CPD33 homolog in Arabidopsis, QUIRKY, had a similar carbohydrate hyperaccumulation phenotype. Radioactively labeled sucrose transport assays showed that sucrose export was significantly lower in cpd33 mutant leaves relative to wild-type leaves. However, PD transport in the adaxial-abaxial direction was unaffected in cpd33 mutant leaves. Intriguingly, transmission electron microscopy revealed fewer PD at the companion cell-sieve element interface in mutant phloem tissue, providing a possible explanation for the reduced sucrose export in mutant leaves. Collectively, our results suggest that CPD33 functions to promote symplastic transport into sieve elements.

Long-Term Outcomes of a Multimodal Day-Clinic Treatment for Chronic Pain under the Conditions of Routine Care.

Chronic pain has high prevalence rates and is one of the top causes of years lived with disability. The aim of the present study was to evaluate the long-term effects of a multimodal day-clinic treatment for chronic pain. The sample included 183 chronic pain patients (114 females and 69 males; 53.3 ± 9.8 years) who participated in a four-week multimodal day-clinic treatment for chronic pain. The patients' average current pain intensity (NRS), sensory and affective pain (Pain Perception Scale), and depression and anxiety (HADS) were assessed at pre- and posttreatment, as well as at three follow-ups (one month, six months, and twelve months after completion of the treatment). Multilevel models for discontinuous change were performed to evaluate the change of the outcome variables. Improvements from pretreatment to posttreatment and from pretreatment to all follow-ups emerged for pain intensity (NRS; 0.54 ≤ d ≤ 0.74), affective pain (Pain Perception Scale; 0.24 ≤ d ≤ 0.47), depression (HADS; 0.38 ≤ d ≤ 0.53), and anxiety (HADS; 0.26 ≤ d ≤ 0.43) (all p < 0.05). Sensory pain as assessed with the Pain Perception Scale did not show any significant change. Patients suffering from chronic pain benefited from the multimodal pain treatment up to twelve months after completion of the treatment.

Exploring genetic variation in the tomato (Solanum section Lycopersicon) clade by whole-genome sequencing.

We explored genetic variation by sequencing a selection of 84 tomato accessions and related wild species representative of the Lycopersicon, Arcanum, Eriopersicon and Neolycopersicon groups, which has yielded a huge amount of precious data on sequence diversity in the tomato clade. Three new reference genomes were reconstructed to support our comparative genome analyses. Comparative sequence alignment revealed group-, species- and accession-specific polymorphisms, explaining characteristic fruit traits and growth habits in the various cultivars. Using gene models from the annotated Heinz 1706 reference genome, we observed differences in the ratio between non-synonymous and synonymous SNPs (dN/dS) in fruit diversification and plant growth genes compared to a random set of genes, indicating positive selection and differences in selection pressure between crop accessions and wild species. In wild species, the number of single-nucleotide polymorphisms (SNPs) exceeds 10 million, i.e. 20-fold higher than found in most of the crop accessions, indicating dramatic genetic erosion of crop and heirloom tomatoes. In addition, the highest levels of heterozygosity were found for allogamous self-incompatible wild species, while facultative and autogamous self-compatible species display a lower heterozygosity level. Using whole-genome SNP information for maximum-likelihood analysis, we achieved complete tree resolution, whereas maximum-likelihood trees based on SNPs from ten fruit and growth genes show incomplete resolution for the crop accessions, partly due to the effect of heterozygous SNPs. Finally, results suggest that phylogenetic relationships are correlated with habitat, indicating the occurrence of geographical races within these groups, which is of practical importance for Solanum genome evolution studies.

Parvoviruses cause nuclear envelope breakdown by activating key enzymes of mitosis.

Disassembly of the nuclear lamina is essential in mitosis and apoptosis requiring multiple coordinated enzymatic activities in nucleus and cytoplasm. Activation and coordination of the different activities is poorly understood and moreover complicated as some factors translocate between cytoplasm and nucleus in preparatory phases. Here we used the ability of parvoviruses to induce nuclear membrane breakdown to understand the triggers of key mitotic enzymes. Nuclear envelope disintegration was shown upon infection, microinjection but also upon their application to permeabilized cells. The latter technique also showed that nuclear envelope disintegration was independent upon soluble cytoplasmic factors. Using time-lapse microscopy, we observed that nuclear disassembly exhibited mitosis-like kinetics and occurred suddenly, implying a catastrophic event irrespective of cell- or type of parvovirus used. Analyzing the order of the processes allowed us to propose a model starting with direct binding of parvoviruses to distinct proteins of the nuclear pore causing structural rearrangement of the parvoviruses. The resulting exposure of domains comprising amphipathic helices was required for nuclear envelope disintegration, which comprised disruption of inner and outer nuclear membrane as shown by electron microscopy. Consistent with Ca⁺⁺ efflux from the lumen between inner and outer nuclear membrane we found that Ca⁺⁺ was essential for nuclear disassembly by activating PKC. PKC activation then triggered activation of cdk-2, which became further activated by caspase-3. Collectively our study shows a unique interaction of a virus with the nuclear envelope, provides evidence that a nuclear pool of executing enzymes is sufficient for nuclear disassembly in quiescent cells, and demonstrates that nuclear disassembly can be uncoupled from initial phases of mitosis.

Isolation and characterization of pathogen-bearing endosomes enable analysis of endosomal escape and identification of new cellular cofactors of infection.

Many pathogens, including viruses, bacteria, as well as bacterial toxins, enter their target cells by endocytosis leading to accumulation of pathogenic and cellular proteins in endosomes. Here, we present detailed experimental instructions on isolation of endosomes after virus infection and their subsequent biomolecular characterization. The isolation of endosomes is based on discontinuous sucrose gradient centrifugation, where different endosomal compartments accumulate at a specific sucrose interface. This enables the enrichment and separation of the virus-interacting and co-internalized cell-surface receptors and membrane-associated proteins. The endosomal fractions can be further analyzed by Western blot or quantitative real-time PCR, which reveals changes in the viral protein or DNA content during the processes of endocytosis and endosomal escape. In addition, comparative quantitative mass spectrometry enables the identification of unknown host-cell factors required for infection.

Epigenetic and genetic influences on DNA methylation variation in maize populations.

DNA methylation is a chromatin modification that is frequently associated with epigenetic regulation in plants and mammals. However, genetic changes such as transposon insertions can also lead to changes in DNA methylation. Genome-wide profiles of DNA methylation for 20 maize (Zea mays) inbred lines were used to discover differentially methylated regions (DMRs). The methylation level for each of these DMRs was also assayed in 31 additional maize or teosinte genotypes, resulting in the discovery of 1966 common DMRs and 1754 rare DMRs. Analysis of recombinant inbred lines provides evidence that the majority of DMRs are heritable. A local association scan found that nearly half of the DMRs with common variation are significantly associated with single nucleotide polymorphisms found within or near the DMR. Many of the DMRs that are significantly associated with local genetic variation are found near transposable elements that may contribute to the variation in DNA methylation. Analysis of gene expression in the same samples used for DNA methylation profiling identified over 300 genes with expression patterns that are significantly associated with DNA methylation variation. Collectively, our results suggest that DNA methylation variation is influenced by genetic and epigenetic changes that are often stably inherited and can influence the expression of nearby genes.

Sorafenib in combination with intensive chemotherapy in elderly patients with acute myeloid leukemia: results from a randomized, placebo-controlled trial.

PURPOSE: The prognosis of elderly patients with acute myeloid leukemia (AML) is still dismal even with intensive chemotherapy. In this trial, we compared the antileukemic activity of standard induction and consolidation therapy with or without the addition of the kinase inhibitor sorafenib in elderly patients with AML. PATIENTS AND METHODS: All patients received standard cytarabine and daunorubicin induction (7+3 regimen) and up to two cycles of intermediate-dose cytarabine consolidation. Two hundred one patients were equally randomly assigned to receive either sorafenib or placebo between the chemotherapy cycles and subsequently for up to 1 year after the beginning of therapy. The primary objective was to test for an improvement in event-free survival (EFS). Overall survival (OS), complete remission (CR) rate, tolerability, and several predefined subgroup analyses were among the secondary objectives. RESULTS: Age, sex, CR and early death (ED) probability, and prognostic factors were balanced between both study arms. Treatment in the sorafenib arm did not result in significant improvement in EFS or OS. This was also true for subgroup analyses, including the subgroup positive for FLT3 internal tandem duplications. Results of induction therapy were worse in the sorafenib arm, with higher treatment-related mortality and lower CR rates. More adverse effects occurred during induction therapy in the sorafenib arm, and patients in this arm received less consolidation chemotherapy as a result of higher induction toxicity. CONCLUSION: In conclusion, combination of standard induction and consolidation therapy with sorafenib in the schedule investigated in our trial is not beneficial for elderly patients with AML.

Properties of the adeno-associated virus assembly-activating protein.

Adeno-associated virus (AAV) capsid assembly requires expression of the assembly-activating protein (AAP) together with capsid proteins VP1, VP2, and VP3. AAP is encoded by an alternative open reading frame of the cap gene. Sequence analysis and site-directed mutagenesis revealed that AAP contains two hydrophobic domains in the N-terminal part of the molecule that are essential for its assembly-promoting activity. Mutation of these sequences reduced the interaction of AAP with the capsid proteins. Deletions and a point mutation in the capsid protein C terminus also abolished capsid assembly and strongly reduced the interaction with AAP. Interpretation of these observations on a structural basis suggests an interaction of AAP with the VP C terminus, which forms the capsid protein interface at the 2-fold symmetry axis. This interpretation is supported by a decrease in the interaction of monoclonal antibody B1 with VP3 under nondenaturing conditions in the presence of AAP, indicative of steric hindrance of B1 binding to its C-terminal epitope by AAP. In addition, AAP forms high-molecular-weight oligomers and changes the conformation of nonassembled VP molecules as detected by conformation-sensitive monoclonal antibodies A20 and C37. Combined, these observations suggest a possible scaffolding activity of AAP in the AAV capsid assembly reaction.

Reshaping of the maize transcriptome by domestication.

Through domestication, humans have substantially altered the morphology of Zea mays ssp. parviglumis (teosinte) into the currently recognizable maize. This system serves as a model for studying adaptation, genome evolution, and the genetics and evolution of complex traits. To examine how domestication has reshaped the transcriptome of maize seedlings, we used expression profiling of 18,242 genes for 38 diverse maize genotypes and 24 teosinte genotypes. We detected evidence for more than 600 genes having significantly different expression levels in maize compared with teosinte. Moreover, more than 1,100 genes showed significantly altered coexpression profiles, reflective of substantial rewiring of the transcriptome since domestication. The genes with altered expression show a significant enrichment for genes previously identified through population genetic analyses as likely targets of selection during maize domestication and improvement; 46 genes previously identified as putative targets of selection also exhibit altered expression levels and coexpression relationships. We also identified 45 genes with altered, primarily higher, expression in inbred relative to outcrossed teosinte. These genes are enriched for functions related to biotic stress and may reflect responses to the effects of inbreeding. This study not only documents alterations in the maize transcriptome following domestication, identifying several genes that may have contributed to the evolution of maize, but highlights the complementary information that can be gained by combining gene expression with population genetic analyses.

Impact of capsid modifications by selected peptide ligands on recombinant adeno-associated virus serotype 2-mediated gene transduction.

Vectors based on adeno-associated virus serotype 2 (AAV2) belong to today's most promising and most frequently used viral vectors in human gene therapy. Like in many other vector systems, the broad but non-specific tropism limits their use for certain cell types or tissues. One approach to screen for transduction-improved vectors is the selection of random peptide libraries displayed directly on the AAV2 capsid. Although the AAV2 library system has been widely applied for the successful selection of improved gene therapy vectors, it remains unknown which steps of the transduction process are most affected and therefore critical for the selection of targeting peptides. Attachment to the cell surface is the first essential step of AAV-mediated gene transduction; however, our experiments challenge the conventional belief that enhanced gene transfer is equivalent to more efficient cell binding of recombinant AAV2 vectors. A comparison of the various steps of gene transfer by vectors carrying a wild-type AAV2 capsid or displaying two exemplary peptide ligands selected from AAV2 random libraries on different human tumour cell lines demonstrated strong alterations in cell binding, cellular uptake, as well as intracellular processing of these vectors. Combined, our results suggest that entry and post-entry events are decisive for the selection of the peptides NDVRSAN and GPQGKNS rather than their cell binding efficiency.

Changes in genome content generated via segregation of non-allelic homologs.

A careful analysis of two maize recombinant inbred lines (RILs) relative to their inbred parents revealed the presence of several hundred apparently de novo copy number variants (CNVs). These changes in genome content were validated via both PCR and whole exome-array capture-and-sequencing experiments. One hundred and eighty-five genomic regions, which overlap with 38 high-confidence genes, exhibited apparently de novo copy number variation (CNV) in these two RILs and in many instances the same apparently de novo CNV events were observed in multiple RILs. Further analyses revealed that these recurrent apparently de novo CNVs were caused by segregation of single-copy homologous sequences that are located in non-allelic positions in the two parental inbred lines. F(1) individuals derived from these inbred lines will be hemizygous for each of these non-allelic homologs but RIL genotypes will contain these sequences at zero, one or two genomic loci. Hence, the segregation of non-allelic homologs may contribute to transgressive segregation. Indeed, statistical associations between phenotypic quantitative trait loci and genomic losses were observed for two of 14 tested pairs of non-allelic homologs.

Impact of VP1-specific protein sequence motifs on adeno-associated virus type 2 intracellular trafficking and nuclear entry.

Adeno-associated virus type 2 (AAV2) has gained much interest as a gene delivery vector. A hallmark of AAV2-mediated gene transfer is an intracellular conformational change of the virus capsid, leading to the exposure of infection-relevant protein domains. These protein domains, which are located on the N-terminal portion of the structural proteins VP1 and VP2, include a catalytic phospholipase A(2) domain and three clusters of basic amino acids. We have identified additional protein sequence motifs located on the VP1/2 N terminus that also proved to be obligatory for virus infectivity. These motifs include signals that are known to be involved in protein interaction, endosomal sorting and signal transduction in eukaryotic cells. Among different AAV serotypes they are highly conserved and mutation of critical amino acids of the respective motifs led to a severe infection-deficient phenotype. In particular, mutation of a YXXQ-sequence motif significantly reduced accumulation of virus capsids around the nucleus in comparison to wild-type AAV2. Interestingly, intracellular trafficking of AAV2 was shown to be independent of PLA(2) activity. Moreover, mutation of three PDZ-binding motifs, which are located consecutively at the very tip of the VP1 N terminus, revealed a nuclear transport-defective phenotype, suggesting a role in nuclear uptake of the virus through an as-yet-unknown mechanism.

Nuclear translocation of adeno-associated virus type 2 capsid proteins for virion assembly.

Adeno-associated virus (AAV) capsid assembly occurs in the nucleus. Newly synthesized capsid proteins VP1, VP2 and VP3 contain several basic regions (BRs), which may act as nuclear localization signals (NLSs). Mutation of BR2 and BR3, located at the VP1 and VP2 N termini, marginally reduced nuclear uptake of VP1 or VP2, but not of VP3, when expressed in the context of the whole AAV type 2 (AAV2) genome. Combined mutation of BR1, BR2 and BR3 resulted in capsids with slightly reduced amounts of VP1. Expression of isolated VP1/2 N termini revealed an influence of BR3 on nuclear transport, whilst BR1 or BR2 had no effect. However, deletion of an N-terminal fragment in front of the BR elements strongly reduced nuclear uptake of VP1/2 N termini. Mutation of BR4, present in all three capsid proteins, led to their retention in the cytoplasm and to the formation of speckles, resulting in a lack of capsid formation and a significant reduction in VP levels. In a VP fragment comprising BR2, BR3 and BR4, the BR4 element was not necessary for nuclear localization. Mutation of BR5 in the C-terminal part of the VPs resulted in a speckled protein distribution in the nucleus, strongly reduced capsid assembly, and low VP1 and VP2 levels. Taken together, these results showed that BR2 and BR3 have a weak influence on nuclear transport of VP1 and VP2, whilst combined mutation of BR1, BR2 and BR3 influences the stoichiometry of VPs in assembled capsids. BR4 and BR5 play a crucial role in capsid assembly but have no NLS activity.

Comparative population genomics of maize domestication and improvement.

Domestication and plant breeding are ongoing 10,000-year-old evolutionary experiments that have radically altered wild species to meet human needs. Maize has undergone a particularly striking transformation. Researchers have sought for decades to identify the genes underlying maize evolution, but these efforts have been limited in scope. Here, we report a comprehensive assessment of the evolution of modern maize based on the genome-wide resequencing of 75 wild, landrace and improved maize lines. We find evidence of recovery of diversity after domestication, likely introgression from wild relatives, and evidence for stronger selection during domestication than improvement. We identify a number of genes with stronger signals of selection than those previously shown to underlie major morphological changes. Finally, through transcriptome-wide analysis of gene expression, we find evidence both consistent with removal of cis-acting variation during maize domestication and improvement and suggestive of modern breeding having increased dominance in expression while targeting highly expressed genes.

Mapping a neutralizing epitope onto the capsid of adeno-associated virus serotype 8.

Adeno-associated viruses (AAVs) are small single-stranded DNA viruses that can package and deliver nongenomic DNA for therapeutic gene delivery. AAV8, a liver-tropic vector, has shown great promise for the treatment of hemophilia A and B. However, as with other AAV vectors, host anti-capsid immune responses are a deterrent to therapeutic success. To characterize the antigenic structure of this vector, cryo-electron microscopy and image reconstruction (cryo-reconstruction) combined with molecular genetics, biochemistry, and in vivo approaches were used to define an antigenic epitope on the AAV8 capsid surface for a neutralizing monoclonal antibody, ADK8. Docking of the crystal structures of AAV8 and a generic Fab into the cryo-reconstruction for the AAV8-ADK8 complex identified a footprint on the prominent protrusions that flank the 3-fold axes of the icosahedrally symmetric capsid. Mutagenesis and cell-binding studies, along with in vitro and in vivo transduction assays, showed that the major ADK8 epitope is formed by an AAV variable region, VRVIII (amino acids 586 to 591 [AAV8 VP1 numbering]), which lies on the surface of the protrusions facing the 3-fold axis. This region plays a role in AAV2 and AAV8 cellular transduction. Coincidently, cell binding and trafficking assays indicate that ADK8 affects a postentry step required for successful virus trafficking to the nucleus, suggesting a probable mechanism of neutralization. This structure-directed strategy for characterizing the antigenic regions of AAVs can thus generate useful information to help re-engineer vectors that escape host neutralization and are hence more efficacious.